A New Spiroaxane Sesquiterpene from Cultures of the Basidiomycete Pholiota adiposa

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A New Spiroaxane Sesquiterpene from Cultures of the Basidiomycete Pholiota adiposa

Dong-Ze Liuâ,b, Rui-Rui Jiaâ, Fei Wangâ, and Ji-Kai Liuâ

aState Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, China

bGraduate School of Chinese Academy of Sciences, Beijing 100049, China

Reprint requests to Prof. Dr. Ji-Kai Liu. Fax: +86-871-5150227. E-mail: jkliu@mail.kib.ac.cn Z. Naturforsch.2008,63b,111 – 113; received July 20, 2007

A new spiroaxane sesquiterpene (15-hydroxy-6α,12-epoxy-7β,10αH,11βH-spiroax-4-ene,1) was isolated from the culture broth of the basidiomycetePholiota adiposa. The structure of1was established on the basis of MS, IR, ID, and 2D NMR experiments.

Key words:Spiroaxane Sesquiterpene, Culture Broth,Pholiota adiposa, Basidiomycete

Introduction

Pholiota adiposais an edible and medicinal fungus, which has been found to show antimicrobial activities and high inhibition to Sarcoma-180 and E-cancer in mice, and can also prevent infection fromStaphylococ- cus aureus,Escherichia coli andMycobacterium tu- berculosis[1]. So far, some chemical constituents have been reported [2, 3]. As a part of our search for natu- rally occurring bioactive metabolites from higher fungi in China [4 – 7], we have carried out a chemical inves- tigation on the cultures ofP. adiposawhich led to the isolation of a new spiroaxane sesquiterpene (1). This report deals with the isolation and structure elucidation of1.

Experimental Section General experimental procedures

Optical rotation was measured on a Horiba SEPA-300 po- larimeter. The IR spectrum was obtained on a Bruker Tensor 27 instrument with KBr pellets. NMR spectra were recorded on Bruker AM-400 and Bruker DRX-500 spectrometers in CD₃OD and CDCl₃with TMS as an internal standard. The EI-MS was recorded with a VG Autospec-3000 spectrometer. The HRESI-MS was recorded with an API QSTAR Pul- sar 1 spectrometer. Silica gel (200 – 300 mesh, Qingdao Ma- rine Chemical Inc., China) and Sephadex LH-20 (Amersham Biosciences, Sweden) were used for column chromatogra- phy. Fractions were monitored by TLC, and spots were visu- alized by heating silica gel plates sprayed with 10 % H2SO4

in ethanol.

The basidiomycete P. adiposa was collected at Ailao Mountain of Yunnan Province, China, in July 2003 and identified by Prof. Mu Zang, Kunming Institute of Botany, Chinese Academy of Sciences (CAS). Voucher specimens were deposited in the Herbarium of the Kunming Institute of Botany, CAS.

Fermentation and isolation

The culture medium consisted of potato (peel off) (200 g), glucose (20 g), KH2PO4 (3 g), MgSO4 (1.5 g), citric acid (0.1 g), and thiamin hydrochloride (10 mg) in 1 L of deion- ized water. Reagent bottles were used as flask (size: 500 mL;

volume of media: 300 mL). The pH was adjusted to 6.5 be- fore autoclaving. Fermentation was carried out on a shaker at 22^◦C and 150 rpm for 10 days.

The whole culture broth ofP. adiposa(12 L) was filtered and then extracted twice with EtOAc. The organic layer was concentrated in vacuo to give 2.3 g of extract which was chromatographed on a silica gel column and eluted step- wise with CHCl₃-MeOH. Fr. II (7.6 mg), eluted with CHCl₃- MeOH (100 : 1, v/v), was further purified repeatedly over a Sephadex LH-20 column eluting with MeOH to give com- pound1(2.5 mg).

Identification

15-Hydroxy-6α,12-epoxy-7β,10αH,11βH-spiroax-4- ene (1), colorless oil. –[α]²⁹_D^.⁷=−3.85 (c= 0.4, MeOH). – IR (KBr):ν = 3424, 2956, 2927, 2869, 1640, 1460, 1379, 1027 cm⁻¹. –¹H NMR (CD3OD) and¹³C NMR (CD3OD and CDCl₃): see Table 1. – EI MS (70 eV):m/z(%) = 236 (4) [M]⁺, 222 (2), 219 (13), 203 (7), 175 (18), 163 (9), 159

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112 D.-Z. Liuet al.·New Spiroaxane Sesquiterpene from Cultures of the BasidiomycetePholiota adiposa Table 1. NMR spectral data for compounds1and2^a.

Position 1(CD3OD) 1(CDCl3) 2(CDCl3)

1H ¹³C ¹³C ¹³C

1 57.0 55.7 56.0

2 1.99 (m) 33.7 32.4 32.9

1.83 (m)

3 2.30 (m) 33.2 32.4 36.7

4 148.3 146.6 142.8

5 5.47 (s) 127.0 126.3 126.0

6 3.56 (d, 3.0) 89.4 87.6 88.0

7 1.95 (m) 41.4 40.1 40.1

8 1.47 (m) 23.0 21.9 21.9

1.21 (br d, 3.0)

9 1.45 (m) 31.7 30.6 30.6

1.23 (br d, 13.0)

10 1.67 (m) 36.1 34.6 34.7

11 2.42 (m) 38.9 37.7 37.7

12 3.90 (dd, 10.0, 8.0) 72.6 71.6 71.6

3.40 (dd, 8.0, 8.0)

13 0.96 (d, 6.9) 12.0 11.8 11.8

14 0.81 (d, 6.9) 16.8 16.3 16.3

15 4.10 (s) 62.1 62.2 16.8

aChemical shift valuesδ in ppm, coupling constantsJin Hz (in parentheses).

(22), 121 (30), 105 (100), 91 (73). – HRESI-MS:m/z = 259.1675 (calcd. 259.1673 for C15H24O2Na, [M+Na]⁺).

Results and Discussion

Compound1 was obtained as an oil. Its molecu- lar formula of C₁₅H₂₄O₂ was established on the basis of positive FAB-MS,¹³C NMR and DEPT spectra and further confirmed by HRESI-MS. Thus, the structure of1contains four degrees of unsaturation. The IR spectrum indicated the presence of a hydroxy group at 3424 cm⁻¹and a C=C double bond at 1640 cm⁻¹. The

1H NMR spectrum (Table 1) exhibited two secondary methyls atδ ^{= 0.96 (d,}^J= 6.9 Hz, 3H) and 0.81 (d,J= 6.9 Hz, 3H). The¹³C NMR and DEPT spectra (Table 1, in CD₃OD) revealed the presence of twosp²carbons atδ = 148.3 (C-4) and 127.0 (C-5), one oxygenated methine carbon atδ = 89.4 (C-6) and two oxygenated methylene carbons atδ = 72.6 (C-12) and 62.1 (C-15), as well as two methyl groups atδ = 16.8 (C-14) and 12.0 (C-13), four methylenes atδ = 33.7 (C-2), 33.2 (C-3), 31.7 (C-9) and 23.0 (C-8), three methines atδ⁼ 41.4 (C-7), 38.9 (C-11) and 36.1 (C-10), and one qua- ternary carbon atom atδ = 57.0 (C-1).

Fig. 1. The structures of compounds1and2.

Fig. 2. Key HMBC and ROESY correlations of compound1. Comparison of the¹³C NMR data of1 with those of2 which was isolated from the unpolar part of the Haitian Vetiver Oil (Fig. 1) implied that they shared the same planar structure except for the hydroxyl sub- stituent at C-15 [8], causing the downfield shifts of C-4 and C-15 fromδ = 142.8 and 16.8 in2 toδ ^{= 146.6} and 62.2 in1, respectively. The above assignment was further supported by cross peaks between H-15 (δ ⁼ 4.10, s) and C-3, C-4, and C-5 in the HMBC spectrum of1. The relative configurations of 1 were de- termined by comparison with 2 and confirmed by a ROESY experiment. The ROESY correlations (Fig. 2) of 5-H (δ = 5.47, s) with 6-H_eq(δ^{= 3.56, d,}^J^{= 3.0 Hz)} and 7-H_ax (δ = 1.95, m), 6-H_eq with 11-H (δ ^{= 2.42,} m) and 2-Hβ ⁽δ = 1.99, m), and 10-Me (δ ^{= 0.81, d,} J = 6.9 Hz) with 2-Hα ⁽δ = 1.83, m) indicated that H-6, H-7, H-11, and H-10 possessedβ^-,β^-,β^{-, and}α^- orientations, respectively. The important NOE between 5-H and 7-H_ax made the stereochemistry at the spiro center and the configuration at C-7 obvious. On the basis of the evidence mentioned above, the structure of1 was therefore deduced to be 15-hydroxy-6α^,12-epoxy- 7β^,10α^{H, 11}βH-spiroax-4-ene.

Acknowledgement

This project was supported by the Chinese Academy of Sciences (KSCX1-YW-R- 24; KSCX2-YW-G-025).

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